Monoclonal antibodiesMonoclonal antibodies

- products of a single B-lymphocyte clone- homogeneous (antigene-specificity, affinity, isotype)

- in human body: only under pathological circumstances e.g. in gammopathy (malign growth of a certain plasma-cell clone)

-their advantage versus polyclonal antibodies: antibodies of the given specificity and isotype can be produced in large amount and of the same quality

monoclonal antibody

Polyclonal antibody

only oneB-lymphocyte clone

moreB-lymphocyte clone

Polyclonal antibody Monoclonal antibody (low affinity)

Monoclonal antibody(high affinity)

Number of recognized antigen determinants

several (frequent cross-reactions)

one (but frequent cross-reactions)

mostly one

Specificity polyspecific often polyspecific monospecific

Affinity Varying (diverse antibodies)

low high

Concentration of non-specific

immunoglobulines

high low low

Yield high low low

Cost of preparation low high high

Standardisability Impossible (or uneasy)

easy easy

Amount limited limitless limitless

Applicability method-dependent low excellent

Features of polyclonal and monoclonal antibodiesFeatures of polyclonal and monoclonal antibodies

Procedure of monoclonal antibody Procedure of monoclonal antibody production production

Hybridoma technologyHybridoma technology

- immunisation of a mouse/rat with a specific antigen

-removal of the spleen or lymph nodes of the mouse, homogenisation

- fusion of mouse plasma cells (with spleen origin) + mouse tumor cells (plasmocytoma/myeloma cells with B-cell origin)

- identification of antibody producing clones. The newly formed hybridomas are proliferating continuously and producing antibodies which concentrate in the medium.

SpleenImmunisation

Myeloma cellHGPRT-

B cells,HGPRT+

PEG fusion

HAT selection

Testing supernatants for specific antibody production

Procedure of monoclonal antibody production II.Procedure of monoclonal antibody production II.

Selection of hybridoma cells

*Hypoxantine-guanine phosphoribosyltransferase

*

HAT= hypoxanthine, aminopterine, thymidine

aminopterine

(1)Immunisation of a mouse(2) Isolation of B cells from the spleen(3) Cultivation of myeloma cells(4) Fusion of myeloma and B cells(5) Separation of cell lines(6) Screening of suitable cell lines(7) in vitro (a) or in vivo (b) multiplication(8) Harvesting

1. Activation of antigen-specific B-lymphocytes- mouse: inbred lines with characterized genetic information, small amounts of antigen is required for immunisation

The followings influence the efficacy of hybridoma cell separation: a) the way of immunisation (using adjuvants, place of injection: intraperitoneal, foot,

tail-vein (caudal vein), spleen) b) number of repeated shots to reinforce (boost) the immune response c) number of days elapsed between the last vaccination and the fusion (2-4 days)

2. Fusion partners- Sp2/0-Ag14 tetraploid,

non-antibody producing plasmocytoma cells from BALB/c mice

(these plasmocytoma cells have HGPRT /hypoxantine-guanine- phosphoribosyl-transferase/ and thymidine kinase deficiency)

Factors influencing the efficacy of monoclonal antibody production Factors influencing the efficacy of monoclonal antibody production

Possible use of monoclonal antibodiesPossible use of monoclonal antibodies- Identifying cell types

Immunohistochemistry

Characterization of lymphomas with CD (cluster of differentiation) markers

- Isolation of cells

Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral

blood!)

- Blood group determination (with anti-A, anti-B and anti-D monoclonals)

-Analysis of a mixture of antigens

- Identification of cell surface and intracellular antigens

Investigation of T-cell activation

- Targeted chemotherapy

CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma

Prevention of organ rejection after transplantation targeting T cells (anti-T cell

monoclonals)

- Drug elimination with antibodies

Anti-digoxin antibodies for the treatment of digoxin-intoxication

Monoclonal antibodies as drugs?Monoclonal antibodies as drugs?

In immunized (with human antigens) mice the produced antibodies will contain mouse-specific proteins, and therefore, they will elicit an immune response upon administering in human subjects.

(see immunogenicity-determining factors!)

How we can solve this problem?

Evolution of monoclonal antibodies

Mouse

Chimeric

Human

Humanized

Humanizing monoclonal antibodiesHumanizing monoclonal antibodies

1. CAMPATH-1H anti-CD52 monoclonal rat antibody

Repeated treatments with non-human antibodies induced strong immune response in patients. (Problem: HAMA = human anti-mouse antibodies)

The antigen binding region of the rat antibody is exchanged with the antigen binding region of a human antibody – in the resulting antibody, only the CDR will be rat protein (chimeric antibodies).

(Problem: HACA = human anti-chimera antibodies, although they are less immunogenic)

2. In vitro phage display: recombinated VDJ regions are expressed on the surface of the filamentous phage capsid. Then, these phages with high-affinity for a given antigen are separated by affinity chromatography. DNA (encoding the human immunoglobulin genes) from these phages are expressed in transgenic mice.

3. Transgenic mice producing human antibodies: Human germ line (not recombinated) Ig locus can be expressed as a transgene in knock-out mice where the Ig genes of the mice were inactivated. Therefore, human Ig gene recombination occurs in the mouse. Hybridomas can be made from mouse B cells producing human antibodies, thus large amount of monoclonal antibodies can be prepared.

In vitro In vitro phage displayphage display

Selective binding to antigen

Washing

Amplification

Expressed antibodies on the

surface of the phage

Characterization (Separation of DNA segments encoding human

Igs and multiplicate them in transgene mice)

Gene3

Expressed human antibody

Human genes encoding antibodies

Phage display vector

Recombination of VDJ gene

Discard

Recovery

mouse Ig „knock-out” transgene mouse

(transferring genes encoding human Ig)

VDJ átrendeződésa transzgén

egérben

- Tumor therapy

Monoclonals made possible the targeted chemotherapy of tumors. It is cell-type specific, but not specific to malignant cells!)

- Immunsuppressive monoclonals

Cell-type specific immunsuppression

Monoclonal antibodies as drugsMonoclonal antibodies as drugs

Monoclonal antibody nomenclature Monoclonal antibody nomenclature The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary names to a group of medicines called monoclonal antibodies. This scheme is used for the World Health Organization’s International Nonproprietary Names.

Components of nomenclature:

Prefix Target Source Suffix

-ki(n)- interleukin as target -u-human

-ci(r)- cardiovascular -o-mouse

-co(l)- colonic tumor -xi-chimeric

variable

-neu(r)- nervous system Etc.

-zu-humanized Etc.

mab

Example:Abciximabab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody used on the cardiovascular system

Monoclonals in tumor therapyMonoclonals in tumor therapy

1. „Naked MAb”, unconjugated antibodyAnti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphomaAnti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemiaAnti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancerAnti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!)Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. humán!)

2. Conjugated antibodyAnti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin)Anti-CD20 + iodine-131 (tositumomab – Bexxar)

Immunsuppressive monoclonals 1.Immunsuppressive monoclonals 1.

1. Anti-TNF-α antibodiesinfliximab (Remicade): since 1998, chimericadalimumab (Humira): since 2002, recombinant human

2. Etanercept (Enbrel) – dimer fusion protein,TNF-α receptor + Ig Fc-part(Not monoclonal antibody, containing only the Fc part of Ig)

Indications of anti-TNF-α therapy:• Rheumatoid arthritis• Spondylitis ankylopoetica (SPA - M.

Bechterew)• Psoriasis vulgaris, arthritis psoriatica• Crohn-disease, colitis ulcerosa• (usually - still – not in the first line!)

- Muromonab-CD3 (OKT-3) mouse IgG2aAgainst CD3 pan-T-cell antigen, after transplantation; It is rarely (or not) used nowadays (mouse protein!); ongoing trials in diabetes mellitus, with the humanized version

- Omalizumab (Xolair):Anti-IgE humanized IgG1k monoclonalInd.: allergic asthma, Churg-Strauss sy.

- Daclizumab (Zenapax):anti-IL-2 receptor humanized antibodyInd.: transplantation

- basiliximab (Simulect): as daclizumab, but chimeric!

- efalizumab (Raptiva): anti-CD11a, humanized, used in psoriasis

Immunsuppressive monoclonals 2.Immunsuppressive monoclonals 2.

Molecular targeted drugsMolecular targeted drugsName Type Target Indications

Alemtuzumab (Mabcampath)

Daclizumab(Zenapax)

Basiliximab(Simulect)

Rituximab(Rituxan/Mabthera)

Trastuzumab(Herceptin)

Gemtuzumab

Ibritumomab (Y90)

Edrecolomab

Gefitinib

Imatinib

Monoclonal antibody, humanized

Monoclonal IgG1, chimeric

Monoclonal IgG1, chimeric

Monoclonal IgG1, chimeric

Monoclonal IgG1, humanized

Monoclonal IgG4, humanizedCalicheamicinnel konjugált

Monoclonal IgG1, murine

Monoclonal IgG2, murine

EGFR-TKI

KIT-TKI

CD52

IL-2 R

IL-2 R

CD20

HER2/neu

CD33

CD20

EpCAM

EGFR TK

TK

CLL, CML

transplantation

transplantation

Lymphoma

Breast cancer, NSC lung cancer

leukemia

lymphoma

CRC

NSCLC

GIST, CML

RadioimmunotherapyAs Zevalin, Bexxar – monoclonal + isotope

Antibody-directed enzyme prodrug therapy (ADEPT) An enzyme is linked to the antibody, and the enzyme will make citotoxic drug from the later administered prodrug

ImmunoliposomesTargeting nucleotides or drugs in liposomes, linked to an antibody (eg. tumor suppressor gene or tissue-specific gene transfer)

Non-immunological targetsas abciximab (ReoPro): inhibition of thrombocyte-aggregation

Further possibilities with monoclonals